Cellular src gene product detected in the freshwater sponge Spongilla lacustris.

Serum from Rous sarcoma virus tumor-bearing rabbits immunoprecipitated from extracts of the freshwater sponge Spongilla lacustris a tyrosine-specific protein kinase with characteristics similar to the chicken pp60c-src kinase activity. An immune competition assay confirmed the relationship between the protein from sponges and viral pp60v-src.

Expression of the c-src protooncogene in human skin tumors.

Retroviral oncogenes are genetic elements, the expression of which is responsible for the transformed phenotype of cells. These genes are derived from normal cellular DNA sequences called cellular protooncogenes, which are present in all human cells and seem to have potential transforming ability in tumors of nonviral origin, since it is possible that they undergo structural alterations and/or changes in their expression. Human skin tumors were analyzed in this study with respect to the expression of the c-src protooncogene, the cellular homologue of the Rous sarcoma virus transforming gene, by measuring the enzymatic activity of its gene product, the pp60c-src kinase activity. Tyrosine-specific kinase activity was detected in all skin tumors tested. The expression pattern of the c-src gene product in the melanomas tested was differential and varying kinase levels in different metastases from the same patient were detected. The elevation of kinase activity as compared to normal skin ranged from about 4- to 20-fold.

Correlations of inheritance and expression between a tumor gene and the cellular homolog of the Rous sarcoma virus-transforming gene in Xiphophorus.

Neoplastic transformation of pigment cells in the teleostean fish Xiphophorus is mediated by a cellular oncogene (Tu). Normally. Tu is suppressed by multiple regulating genes (R). Depending on impairment and loss of R genes, Tu is permitted to express itself phenotypically. In the pigment cell system, different degrees of Tu expression lead to small spots of transformed cells or to benign or malignant melanoma. All neoplastic and nonneoplastic cells of all Xiphophorus genotypes tested thus far appear to contain the cellular homolog (c-src) of the avian sarcoma virus oncogene (v-src). The evidence for this stems from the detectability of a Mr 60,000 phosphoprotein with associated kinase activity (pp60c-src) that reacts with antiserum against viral pp60src. We followed the inheritance of Tu (identified by spots and melanomas) compared to the expression of c-src identified by the pp60c-src-associated protein kinase). By quantitative determination of kinase activity in immunoprecipitated pp60c-src from fish showing different degrees of Tu expression, we have investigated whether there exists a correlation between the expression of c-src and Tu. In genotypes with the same genetic background, cells from Tu-containing fish express more pp60c-src than do cells from fish lacking Tu. In genotypes carrying a Tu gene and which show differences in the amount of gene expression due to a different extent of repression by regulating genes, analysis of kinase activity revealed that an increase of Tu expression is correlated with an elevated level of pp60c-src-associated kinase activity. Our findings may indicate that c-src activity in Xiphophorus is modulated by the Tu gene product or that Tu and c-src are regulated coordinately.

Protein kinase activity associated with immunopurified p53 protein.

Enhanced protein phosphorylation seems to be characteristic for cell transformation. Viral or cellular oncogene products which are functionally implicated in cell transformation sometimes activate protein kinases, or they are protein kinases themselves. In the present paper we have shown that a protein kinase activity is tightly associated with immunopurified oncoprotein p53, either uncomplexed or in complex with SV40 large T antigen. Furthermore, we could demonstrate that the protein kinase associated with immunopurified p53 was independent of SV40 large T antigen. p53 in the immunocomplexes served as a substrate for this protein kinase. Phosphoamino acid analysis of in vitro phosphorylated p53 revealed a phosphorylation predominantly on serine residues similar to p53 phosphorylated in vivo. The use of different monoclonal antibodies did not reveal a total inhibition of the protein kinase activity. However, p53 precipitated with monoclonal antibodies which recognize a C-terminal domain, was phosphorylated in vitro to a lesser extent than p53 which was precipitated with monoclonal antibodies that recognize an N-terminal epitope. All subclasses of immunopurified p53 separable by sucrose density gradients or by sequential immunoprecipitation exhibited a protein kinase activity and served as substrates for this protein kinase. Moreover, a protein kinase activity was found to be associated with baculovirus expressed p53 which allows us to attribute this enzymatic activity more directly to p53.

Synaptophysin: a substrate for the protein tyrosine kinase pp60c-src in intact synaptic vesicles.

Expression of pp60c-src, the first well defined proto-oncogene product, is developmentally regulated and tissue-specific, with neuronal tissues displaying high amounts of the c-src encoded pp60c-src kinase activity. In the central nervous system pp60c-src is preferentially expressed in regions characterized by a high content of grey matter and elevated density of nerve terminals. In this study we show for the first time a direct interaction between pp60c-src and synaptophysin as a physiological target protein in neurons by demonstrating that endogenous pp60c-src is able to phosphorylate synaptophysin (p38). p38 is a major constituent of the synaptic vesicle membrane protein and is thought to play a key role in the exocytosis of small synaptic vesicles and possibly small clear vesicles in neuroendocrine cells.

Preferential expression of a pp60c-src related protein tyrosine kinase activity in nerve cells of the early metazoan Hydra (Coelenterates).

It has been suggested that the proto-oncogene c-src plays a functional role in developing neurons, and in the mature nerve cells of higher vertebrates. The coelenterate Hydra represents the most primitive known organism possessing nerve cells. With Southern blot hybridizations we have demonstrated src-related sequences in Hydra. Antisera specific for the c-src gene product (pp60c-src) of birds and mammals precipitate a protein from Hydra cell extracts with a tyrosine-specific protein kinase activity. Studies of tissues and cells fractionated from a temperature sensitive mutant of Hydra which is depleted of interstitial (including nerve) cells at the non-permissive temperature, have indicated the src-like kinase of Hydra to be preferentially expressed in nerve cells. The high conservation of structural features and of the expression pattern indicates a basic function for pp60c-src in neurons.

A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

Multiple src-related kinase genes, srk1-4, in the fresh water sponge Spongilla lacustris.

In one of the simplest metazoan organisms, the sponge Spongilla lacustris, at least four different src-related kinase genes (srk1-4) are expressed, all of which show a high degree of similarity to the c-src genes of vertebrates. Whereas srk2 and srk3 are clearly unrelated at the nucleic acid level, srk1 and srk4 share identical sequences in the 5' parts of their cDNAs. The cloning of several primer extension clones and genomic polymerase chain reaction experiments confirmed the hypothesis of an alternative splicing of tandemly arranged carboxy-terminal parts of srk1 and srk4. The genomic sequence encoding both proteins was found to be interrupted at the splice point by an intron which is located in the same position as one of the introns in the chicken src gene, which is the only gene conserved in invertebrates and vertebrates. All four srk genes are expressed in adult sponges as mRNA transcripts of about 2.2 kb. Tyrosine kinase activity of a src-related kinase could be detected in adult sponges but not in their resting form (gemmulae), and may reflect the activity of the srk protein products. Spongilla lacustris is the simplest organism from which a protein tyrosine kinase gene has been isolated. The presence of at least four such genes in the evolutionary ancient and primitive phylum Porifera suggests that tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms and that their activity may be involved in aggregation and cell-cell recognition.

The TP1 isolate of feline sarcoma virus encodes a fgr-related oncogene lacking gamma actin sequences.

We have isolated a new feline sarcoma virus, TP1-FeSV. The virus encodes a myristilated 83 kD gag-onc fusion protein displaying tyrosine kinase activity. We have established nonproducer cell lines lacking the TP1-FeSV associated helper virus (FeLV) and TP1-FeSV transfected NIH cell lines. Southern Blot analysis of genomic DNA and Northern Blot analysis of RNA isolated from these cell lines revealed that the oncogene of the TP1-FeSV isolate is related to the fgr oncogene of the GR-FeSV, but shows no hybridization to the gamma actin homologous sequences of the GR-FeSV. We have isolated TP1-FeSV specific clones from a genomic library. Restriction enzyme and sequence analysis showed that the TP1-FeSV genome consists of the first 1651 nucleotides of the gag gene, followed directly by fgr sequences. The TP1-FeSV fgr sequence starts 43 nucleotides after the beginning of the GR-FeSV fgr sequence. In contrast to the GR-FeSV fgr which has lost 13 amino acids of the c-fgr carboxy terminus, the TP1-FeSV fgr contains the complete carboxy terminus of the cellular fgr gene. The TP1-FeSV fgr sequence is followed by a unique 328 nucleotide long sequence of unknown origin. The 3' recombination site occurs within the pol gene, 460 nucleotides from the start of the env leader sequence. Comparison of the subcellular localization of the transforming proteins of TP1-FeSV and GR-FeSV show no striking difference; both molecules are in part associated with subcellular membrane/cytoskeletal fractions and form complexes with the cellular pp90 and pp50.

Iron uptake studies on erythroid cells.

Iron uptake from 55Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.

Use of iron from transferrin and microbial chelates as substrate for heme synthetase in transformed and primary erythroid cell cultures.

The enzymatic heme production in cell-free extracts of virus-transformed Friend erythroleukemia cells and primary bone marrow cells from rabbits has been measured by determining the activity of heme synthetase after addition of iron sulfate, transferrin or microbial iron chelates. In transformed cells the amounts of heme formed did not show significant difeerences independent of which substrate was offered. In cell-free extracts of primary bone marrow cells no increase of heme production could be observed.

The differential expression of the cellular src-gene product pp60src and its phosphokinase activity in normal chicken cells and tissues.

Chick embryos of different ages and adult chickens were examined for the expression of pp60c -src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. In any brain extract, the pp60c -src kinase activity was always high, whereas muscle extracts of embryos show an age-dependent decrease in kinase activity. Adult animals show either no or barely measurable activity in muscle tissue. In contrast, liver cell extracts of embryos show an age-dependent increase in pp60c -src kinase activity, with adult chickens displaying the highest activity, very similar to that found in brain extracts. This demonstrates that increased expression of c-src is not necessarily correlated with cell proliferation, but suggests that, at an early stage of differentiation of mesenchymal cells, the relatively high expression of c-src could be responsible, at least in part, for the control of cell metabolism and proliferation.

Microbial iron chelates with iron donor properties in hemoglobin-synthesizing cells.

Iron incorporation into Friend virus infected leukemic murine spleen cells was studied using the two fungal iron trihydroxamates, fusigen and ferricrocin. Incorporation of 55Fe was measured by isolation of hemoglobin after dimethylsulfoxide-induced hemoglobin synthesis and compared with iron incorporation from 55Fe-labeled ferric citrate.

Ecto-protein kinase activities in normal and transformed cells.

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5''-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.

Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells.

Small GTP binding proteins of the rab/YPT family are essential regulators of vectorial transport in the eukaryotic cell. Members of the rab/YPT1 family are found on the cytoplasmic surface of distinct intracellular membrane compartments. Membrane attachment is facilitated by a C-terminal geranylgeranyl moiety. In this report we investigated posttranslational modification and membrane binding of the rab6 protein, a member of the rab/YPT family located on the Golgi apparatus. A set of point mutations, which simulate the GDP or GTP bound conformation, was introduced into the rab6 cDNA. The mutated cDNAs were expressed in insect cells and the ability of the protein products to undergo geranylgeranyl modification and membrane association was assessed by Triton X-114 partition and cell fractionation. We report here that the modification of rab6 in insect cells depends on protein conformation. Only the GDP bound form, but not the GTP bound form is isoprenylated and subsequently membrane bound.

Expression analyses of a c-src related gene in sponges.

Applying different hybridization techniques we could confirm existence and transcription of sequences related to the proto-oncogene c-src in freshwater and marine species of sponges, the most primitive metazoan animals. By using the in situ hybridization technique, we were able to demonstrate for the first time that the cell-type-specific expression, characteristic for c-src related genes in higher vertebrates, is even realized in a freshwater sponge (Spongilla lacustris). The highest amount of c-src related transcripts was found in the omnipotent stem cells, the archaeocytes, and in the cells forming the inner epithelia, the endopinacocytes, which probably are involved in signal transduction.

Expression of tyrosine kinases encoding protooncogenes in differentiating human leukemia-cells.

We investigated the expression of the tyrosine kinase encoding proto-oncogenes c-src and fyn during in vitro differentiation of the human promyelocytic leukemia cell line HL-60 and an HL-60 derived cell line (HL-GOT) with an altered T-cell receptor gene. In HL-60 cells, the fyn and c-src encoded tyrosine kinases are activated in a differentiation-dependent manner, whereas in HL-60T cells high levels of pp59(fyn) and pp60(c-src) specific kinase activities are already present in the untreated cells. From the data obtained we conclude that untreated HL-60T cells represent a more differentiated type of cells compared to the untreated promyelocytic HL-60 cells.

Inactive and active mutants of rab1b are not tightly integrated into target membranes.

The rab1b GTPase is localized on the ER and cis-Golgi membranes and involved in early exocytotic membrane traffic processes. To analyze the influence of rab1b conformation on specific membrane targeting processes three point mutants simulating permanently inactive or active rab1b proteins were constructed and expressed in BHK cells. To increase the expression of the growth inhibiting rab1b S22N and rab1b N121I mutants we co-expressed the Mss4 protein and observed an elevated expression of the inactive rab1b mutants. Surprisingly, only the rab1b wild-type protein shows the correct intracellular localization, and a tight membrane association. We conclude that the targeting process of rab1b depends predominantly on GDP/GTP exchange.

A new polyoxometalate complex inhibits retrovirus encoded reverse transcriptase activity in vitro and in vivo.

We examined the recently synthesized and characterized polyoxometalate compound (NH4)10[Co2Sb2W20 O70(H2O)6] (POM1). The inhibitory potency of POM1 was studied in tissue culture experiments with uninfected and Rous sarcoma virus (RSV)-infected chicken embryo fibroblasts (CEF) in vivo. We measured a considerable decrease in total cellular phosphotyrosine content in treated infected cells in vivo. POM1 treatment of SR-RSV-A infected CEF in vivo resulted in decreased pp60v-src activity, possibly due to a reduced rate of v-src translation caused by the inhibitory effect of POM1 on the RSV encoded reverse transcriptase activity (RT) activity. In further studies we were able to demonstrate the inhibitory effect of this complex on the RT activity of RSV in vitro and in vivo.

Competitive-differential polymerase chain reaction for gene dosage estimation of erbB-1 (egfr), erbB-2, and erbB-3 oncogenes.

Increases in the average gene copy number (AGCN) of the erbB oncogenes, especially the erbB-2 gene, have been found in a variety of human cancers. Such information is useful with respect to prognosis of the disease. Poor reproducibility and DNA damage complicate quantitative polymerase chain reaction (PCR)-based methods. Therefore, we describe a quantitative PCR method for the estimation of AGCN in the oncogenes erbB-1 (egfr), erbB-2, and erbB-3. The method comprises a competitive and differential PCR in a one-tube reaction (competitive-differential PCR, or cdPCR). Genomic sequences of the oncogene and the human beta-globin (HBB) reference gene and two competitors for the oncogene and reference gene were amplified with two primer pairs simultaneously. The competitors were chosen to be amplified with the same efficiency as the genomic sequences. Using this method, we confirmed egfr and erbB-2 amplification in cancer cell lines and tumor tissue, and we also detected erbB-3 amplifications. Furthermore, gene dosage decreases were detectable, e.g., an erbB-2 hemizygosity in MCF-7 cells. Thus, cdPCR facilitates the detection of both increases and decreases in AGCN with high reproducibility, sensitivity, and accuracy. This method is therefore suitable for clinical studies on erbB oncogene dosage deviations.